Isolation of Yeast Genomic DNA for Southern Blot Analysis

2006 ◽  
Vol 2006 (1) ◽  
pp. pdb.prot4149 ◽  
Author(s):  
David C. Amberg ◽  
Daniel J. Burke ◽  
Jeffrey N. Strathern
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Genomic Dna ◽  
Southern Blot Analysis

scholarly journals Expression of transcobalamin II mRNA in human tissues and cultured fibroblasts from normal and transcobalamin II-deficient patients

1994 ◽  
Vol 301 (2) ◽  
pp. 585-590 ◽  
Author(s):  
N Li ◽  
S Seetharam ◽  
D S Rosenblatt ◽  
B Seetharam
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Genomic Dna ◽  
Southern Blot Analysis ◽  
Sequence Similarity ◽  
Ribonuclease Protection Assay ◽  
Rat Tissues ◽  
Cultured Fibroblasts ◽  
Ribonuclease Protection ◽  
Transcobalamin Ii

Transcobalamin II (TCII) is an important plasma transporter of cobalamin (Cbl; vitamin B12). In the present study, TCII gene expression in human and rat tissues and in the fibroblasts of patients with TCII deficiency was investigated. Northern-blot analyses revealed expression of TCII mRNA in many human and rat tissues. In humans, this was 14-fold higher in the kidney than in liver, whereas in the rat the levels of expression were similar in the kidney and liver. Southern-blot analysis of genomic DNA from several species revealed sequence similarity in TCII across species. Metabolic labelling and ribonuclease protection assay revealed a 43 kDa TCII protein and a fully protected TCII mRNA band in normal fibroblasts but not in fibroblasts from three TCII-deficient patients. Southern-blot analysis of genomic DNA from all these fibroblasts revealed identical restriction patterns on BamHI, HindIII, KpnI, MspI and EcoRI digestion. On the basis of these results, we suggest that TCII is expressed in multiple tissues, and its level of expression in tissues varies within the same and across species. Furthermore, the TCII deficiency characterized in this study is due to the absence of TCII protein which in turn is due to the absence or extremely low levels of its mRNA and not to detectable gross alterations in the gene structure.

scholarly journals Treatment of genomic DNA with T4 DNA ligase improves Southern blot analysis

1988 ◽  
Vol 16 (21) ◽  
pp. 10387-10387 ◽  
Author(s):  
Jørn Erland Koch ◽  
Steen Kølvraa ◽  
Morten Corneliusen ◽  
Kirsten Brunn Petersen ◽  
Niels Gregersen
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Genomic Dna ◽  
Southern Blot Analysis ◽  
Dna Ligase ◽  
T4 Dna Ligase

scholarly journals Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice [see comments]

Blood ◽  
1993 ◽  
Vol 81 (10) ◽  
pp. 2566-2571 ◽  
Author(s):  
FM Stewart ◽  
RB Crittenden ◽  
PA Lowry ◽  
S Pearson-White ◽  
PJ Quesenberry
Keyword(s):  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Normal Male ◽  
Normal Donor ◽  
Long Term Stability ◽  
Donor Bone Marrow ◽  
Male Donor ◽  
Marrow Cells

We report the successful long-term engraftment of normal male donor bone marrow (BM) transfused into noncytoablated female mice, challenging the assumption that “niches” need to be created for marrow to engraft. We have used chromosomal banding and Southern blot analysis to identify transplanted male marrow cells, and shown the long-term stability of the chimeric marrows. Balb/C, BDF1, or CBA-J female hosts (no irradiation) received for 5 consecutive days 40 x 10(6) male cells (per day) of the same strain, and repopulation patterns were observed. Parallel studies were performed using tibia/femur equivalents of normal marrow or marrow from Balb/C mice pretreated 6 days previously with 150 mg/kg 5-fluorouracil (5-FU). Chromosome banding techniques showed that 5% to 46% of marrow cells were male 3 to 9 months posttransplant with normal donor marrow. Southern blot analysis, using the pY2 probe, showed continued engraftment at 21 to 25 months posttransplant, ranging from 15% to 42% male engrafted cells in marrow. Normal donor male marrow engrafted significantly better than 5-FU-pretreated male marrow as shown 1 to 12 months posttransplant in non-cytoablated female recipients. Percentages of male engrafted cells in BM ranged from 23% to 78% for recipients of normal donor marrow and from 0.1% to 39% for recipients of 5-FU marrow. Mean engraftment for 6 mice receiving normal marrow was 38%, whereas that for 6 mice receiving post-5-FU marrow was 8%, as assayed 1 to 3 months posttransplant. At 10 to 12 months, mean engraftment for the normal donor group was 46%, compared with 16% for the 5-FU group. The patterns of engraftment with normal and 5-FU marrow were similar for spleen and thymus. These results show that long-term chimerism can be established after transplantation of normal donor marrow to normal nonirradiated host mice and indicate that marrow spaces do not have to be created for successful engraftment. They suggest that transplanted marrow competes equally with host marrow for marrow space. Finally, these data show that post-5-FU Balb/C male marrow is markedly inferior in the repopulation of Balb/C female host marrow, spleen, and thymus, and suggest that this population of cells may not be the ideal population for gene transfer studies.

scholarly journals Mutational analysis of the CDKN2 (MTS1/p16ink4A) gene in primary B-cell lymphomas

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2724-2731 ◽  
Author(s):  
T Uchida ◽  
T Watanabe ◽  
T Kinoshita ◽  
T Murate ◽  
H Saito ◽  
...  
Keyword(s):  
B Cell ◽  
Missense Mutation ◽  
Southern Blot ◽  
Blot Analysis ◽  
Southern Blot Analysis ◽  
Mutational Analysis ◽  
Direct Sequencing ◽  
Lymphocytic Leukemia ◽  
Sscp Analysis ◽  
Putative Tumor Suppressor Gene

Abstract The CDKN2 gene located on chromosome 9p21 encodes the cyclin-dependent kinase-4 inhibitor p16. This gene is a putative tumor-suppressor gene because of its frequent alterations in many kinds of tumor cell lines. We analyzed the CDKN2 gene to evaluate its alterations in 52 primary specimens of non-Hodgkin's lymphoma (NHL) or chronic lymphocytic leukemia (CLL) of B-cell origin by Southern blot analysis, polymerase chain reaction-mediated single-strand conformation polymorphism (PCR-SSCP) analysis, and direct sequencing. By Southern blot analysis, we showed homozygous deletion of the CDKN2 gene in 3 of 42 patients with B-NHL (7.1%). After screening by PCR-SSCP analysis, direct sequencing identified one missense mutation at codon 72 (nucleotide 233) and two frameshifts due to a 35-bp deletion arising at codon 49 (nucleotides 163 to 175) in patients with B-NHL (3 of 42, 7.1%). In the patient carrying the missense mutation, hemizygous deletion of the CDKN2 gene was also suspected. In this study, we detected alterations in CDKN2 in 6 of 42 patients (14.3%) with B-NHL and in none of 10 patients with B-CLL. Our results suggest that the CDKN2 alterations contribute in tumorigenesis in some patients with B-NHL.

Sign in / Sign up

Export Citation Format

Share Document